A definitive and generally accepted role for the vitamin D-induced calcium-binding protein (CaBP) is not available at the present time despite the fact that more than 10 years have elapsed since its discovery. The results of research in the intervening years have suggested an involvement of the protein in vitamin D-dependent calcium metabolism, but, an understanding of the exact mechanism of that role suffers to a large degree from the controversy concerning the immunocytological localization of CaBP. The proposed investigations are designed to systematically investigate the variables in the protocols which have been used in previous localization studies with the goal of identifying the factor(s) responsible for the divergent results. With that information, a reliable cytological localization protocol will be designed. The localization of intestinal CaBP will be investigated under different nutritional and physiological conditions known to alter both intestinal calcium transport and CaBP content. CaBP will be localized using the same protocol in two other calcium-transporting organs, the kidney and shell gland of the laying hen. The protein will also be localized in brain. Both the light microscopic and the ultrastructural localization of CaBP will be investigated. Reliable information concerning the CaBP site(s) in these tissues will greatly aid in defining the function(s) of the protein in calcium metabolism which in turn may be of value in gaining a better unerstanding of the causes of altered calcium metabolism in different disease states. A study is also proposed to isolate and purify a mitochondrial calcium binding protein, a glycoprotein (CaBGP), then raise a monospecific antiserum against the protein. The cytological localization of CaBGP will be studied as a function of the mineralization state in calcifying cartilage. The potential involvement of CaBGP in calcification reactions will be investigated.